Fig. 6.

Increased occupancy of MeCP2 and HP1 but not H2A.Z at the Bsp gene in Osx-null calvarial cells. (A–C) ChIP assays for occupancy of MeCP2, HP1, and H2A.Z at chromatin segments of the Bsp gene in Osx-wt (Wt) and Osx-null calvarial cells. ChIP data for MeCP2 and HP1α occupancy are presented as fold enrichment over control rabbit IgG values and for H2A.Z as % bound/input after subtracting control rabbit IgG values. Site-specific primers P2 and P4 are within the promoter and P5+ within the coding region of the Bsp gene. (D) Panel 1, Coimmunoprecipitation of endogenous HP1α in the lysates of Osx-wt calvarial cells with NO66 antibody followed by western blot with HP1α antibody. Panel 2–4, Purification of Flag-NO66 from nuclear extract of Flag-NO66 expressing HEK293 cells with anti-Flag M2 agarose (Sigma) and then elution with 3X Flag peptides, followed by separation of eluate on SDS-PAGE and western blot with antibodies as indicated in the panel. (E) HP1α stimulates H3K4me3 and H3K36me3 demethylation by the NO66 demethylase. The demethylation reaction was carried out with E. coli expressed NO66 (full-length) and HP1α using calf-thymus bulk histones as substrates. Lower amount of NO66 that showed no demethylase activity was used in this reaction (1x equivalent to 1–2 μg). (F) Osx inhibits the NO66-dependent demethylation of H3K4me3 and H3K36me3 in a reaction that contained E. coli-expressed His-NO66 (168–641) and His-Osx (1–225). His-NO66 (168–641) that contained an intact JmjC domain also exhibited demethylase activity. Samples were run on the same gel and relevant lanes were shown (Panel E and F).